Adoptive T cell therapy is a breakthrough technology that utilizes ex vivo propagated T cells genetically modified with T cell receptors or with Chimeric Antigen Receptors (CAR) (Rosenberg, The New England Journal of medicine, 350:1461-1463 (2004)), Kershaw, et al., Nature Review Immunology, 5:928-940 (2005)). Among these methods, CAR-T cell therapy is the most widely applicable since it allows MHC-independent tumor antigen recognition and T cell activation without the need for co-stimulatory signals (Kochenderfer, et al., Nat Rev Clin Oncol, 10:267-276 (2013). The CAR contains an extracellular single-stranded antibody (scFv), a transmembrane anchor, and signaling domains. To date, most CAR-T cell proteins for B-cell malignancies have been designed to recognize CD19, CD20 or ROR1 antigens (Hudecek, et al., An Official Journal of the American Association for Cancer Research, 19:3153-64 (2013)); Till, et al., Blood, 119:3940-3950 (2012); Jensen, et al., Immunol Rev, 257:127-44 (2014)). When expressed on the surface of a T cell, CAR mediates binding to the target tumor antigen and activates T cell cytotoxic response by release of granzymes, perforin and granulysin, as well as expression of Fas ligand, TRAIL and TNF (Kershaw, et al., Nature Reviews Immunology, 5:928-940 (2005)). These proteins induce death of target cells through multiple means, for example, through direct caspase-3/7 activation or by indirect caspase-3/7 activation through cleavage and activation of the BH3-only protein BID by granzyme B or Fas and TNF1 triggered caspase-8 activation (Barry, et al., Molecular and Cellular Biology, 20:3781-3794 (2000); Li, et al., Cell, 94:491-501 (1998); Luo, et al., Cell, 94:481-90 (1998)). Early clinical trials of CD19-CAR T cells have demonstrated remarkable efficacy for chronic lymphocytic leukemia (CLL) (Kalos, et al., Science Translational Medicine, 3:95ra73 (2011)) and acute lymphocytic leukemia (ALL) (Brentjens, et al., Science Translational Medicine, 5:177ra38 (2013)). In addition, limited pre-clinical and clinical success has been shown for MCL with CAR T cells directed against CD20 or CD19 (Hudecek, et al., An Official Journal of the American Association for Cancer Research, 19:3153-64 (2013); Till, et al., Blood, 119:3940-3950 (2012); Koehenderfer, et al., Blood, 122:4129-39 (2013); Till, et al., Blood, 112:2261-2271 (2008)). This approach has been so successful in treating B-Lymphoblastic Leukemia and Chronic Lymphocytic Leukemia, that it has been given special NIH Breakthrough Status (Gill, et al., Immunological Reviews, 263:68-89 (2015)).
However, there are several drawbacks of standard methods that reprogram T cells with CAR DNA. First, the approach does not control the in vivo proliferation of T cells and a common complication in several clinical trials has been severe cytokine storm due to uncontrolled CAR-T cell activity (Kalos, et al., Science Translational Medicine, 3, 95ra73 (2011)). Second, persistent CD19 CAR-T cells can lead to the permanent depletion of B cells and increased susceptibility to infections (Porter, et al., The New England Journal of Medicine, 365:725-733 (2011)). Third, DNA reprogramming requires cells with a high proliferative potential (Jensen, et al., Immunological Reviews, 257: 127-144 (2014); Chen, et al., Immunity, 39:1-10 (2013); Hinrichs, et al., Immunological Reviews 257:56-71 (2014)) which may be difficult to attain especially from some patients. Fourth, CAR T cells infused in patients can be suppressed by the lack of adequate stimulation, and while pre-treatment with lymphotoxic drugs minimizes suppression (Jensen, et al., Immunological Reviews, 257: 127-144 (2014)), it considerably increases the toxicity of the treatment. Finally, introduction of CARs by retroviral transduction carries the risk of insertional mutagenesis and oncogene activation (Hacein-Bey-Abina, et al., The New England Journal of Medicine, 348:255-256 (2003)).
Non-viral methods of gene delivery were initially developed on DNA models and include electroporation, liposomal, polymer, polypeptide dependent delivery and transfection with “naked” DNA. Electroporation utilizes the application of brief, high-voltage electric pulses to a variety of animal and plant cells and leads to the formation disturbances in the plasma membrane (U.S. Pat. No. 4,394,448 to Szoka, Jr., et al. and U.S. Pat. No. 4,619,794 to Hauser). Nucleic acids can enter directly into the cell cytoplasm either through these, or as a consequence of the redistribution of membrane components that accompanies membrane restoration. Liposomal and polypeptide dependent approaches mix the material to be transferred with non-toxic polymers to form particles able to penetrate cells and to deliver nucleic acids into cytoplasm (Feigner and Ringold, Nature, 337:387-388 (1989), Saltzman and Desai, Annals of Biomedical Engineering, 34, 270-275 (2006). Polypeptide dependent approaches involve the use of highly penetrating proteins and peptides mixed with a nucleic acid followed by exposure of a target cell to the nucleoprotein/nucleopeptide complex (Verma and Somia, Nature, 389:239 (1997); Wolff et al., Science, 247:1465 (1990)). “Naked” DNA transfection approaches involve methods where nucleic acids are administered directly in vivo (Herweijer and Wolff, Gene Ther. 10(6):453-8 (2003)).
A common disadvantage to known non-viral DNA delivery techniques is that the amount of exogenous protein expression produced relative to the amount of exogenous nucleic acid administered remains too low for most diagnostic or therapeutic procedures. Low levels of protein expression are often a result of a low rate of transfection of the nucleic acid and/or toxicity of exogenous DNA. In addition, some types of cells are very resistant to DNA transfection, and introduced foreign DNA can incorporate into the genome and act as a mutagen.
An alternative procedure for non-viral gene delivery is achieved by transfection of mRNA rather than DNA. In principle, unlike DNA transfection, introducing mRNA can have no permanent effect on the genetic structure of the cell, at least in the absence of rare reverse transcription events. There is limited literature on the application of mRNA transfection approaches (for example, Seaboe-Larssen, et al., J. Immm. Methods, 259:191-203 (2002); Boczkowski, et al., Cancer Res., 60:1028-1034 (2001); and Elango et al., Biochem. Biophys. Res. Comm., 330, 958-966 (2005)), and little in the way of a systematic comparison of DNA and RNA transfection procedures. Most available literature for mRNA transfection is based on methods that involve labor intensive cloning of the gene of interest in special vectors containing a bacteriophage promoter upstream and polyA/T stretch downstream of the cloning site. Not only is cloning time consuming, but recombinant plasmids containing a stretch of poly(AIT) are often unstable in bacterial cells and prone to spontaneous mutations (Kiyama, et al., Gene, 150:1963-1969 (1994)). Furthermore, most mRNAs that are generated from d(A/T)n vectors contain a short sequence of heterologous nucleotides following the poly(A) tail. The influence of these heterologous sequences on translation is unknown (Elango et al., Biochem. Biophys. Res. Comm., 330, 958-966 (2005)).
Accordingly, there remains a need for improved methods of making RNA in vitro. There also remains a need for improved methods of cell modification that do not rely on genomic integration of the modifying construct.
It is an object of the present invention to provide a more convenient and/or efficient method of mRNA production for transfection of different types of cells, including cells which are not transfectable for DNA.
It is also an object of the present invention to provide a method of mRNA transfection with minimal side effects and high efficiency, which allows transient expression of genes and desirable modification of cell phenotype without causing permanent genetic changes, which avoids risk associated with conventional gene therapy.
It is also an object of the present invention to provide a method of cell transfection with multiple genes wherein the level of each gene expression can be individually controlled.
It is an object of the present invention to provide a method of transfection of primary mammalian cells, including human cells, and use of those cells for therapy of cancer, autoimmune and infectious diseases.
It is an object of the present invention to provide a method of transfection of primary mammalian cells, including human cells, and use of those cells for treatment of a variety of human diseases including neurological diseases, organ regeneration, and restoration of the immune system.
It is another object of the present invention to provide a method of transient cell modification, which allows fast and safe generation of diverse differentiation, de-differentiation, re-differentiation, or reprogramed states of cells of different cell types, including diverse stem cells from various tissues such as fibroblasts, hematopoietic, epithelial cells and others.
It is another object of the invention to provide modified cells and methods of use thereof in various therapeutic strategies to treat a wide range of cancers and other diseases and disorders.
It is a further object of the invention to provided transfected cells that exhibit (1) CAR reprogramming, (2) improved survival and metabolic stability, (3) ability to distinguish tumor from normal cells, (4) ability to avoid immunosuppression, and combinations thereof.